Methods and compositions for inhibiting mutant EGFR signaling

ABSTRACT

Methods of inhibiting mutant EGFR and methods of treating a subject afflicted with a lung cancer having a mutant EGFR, having for example a C797 mutation, are described. The methods comprise administering to a cell or a subject in need thereof a therapeutically effective amount of a compound selected from 3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and a structurally related analog thereof; midostaurin; and AZD7622 and a structurally related analog thereof; and mixtures thereof. Compositions and combinations comprising the compounds of the disclosure as well as uses are also provided.

This application is a divisional of U.S. patent application Ser. No.16/146,611, filed Sep. 28, 2018, which claims the benefit of 35 USC 119based on the priority of U.S. Provisional Application No. 62/564,599filed Sep. 28, 2017, which are herein incorporated by reference.

FIELD

The disclosure relates to methods and compositions for inhibiting mutantEGFR signaling and in particular for inhibiting EGFR triple mutantscomprising a C797 mutation.

BACKGROUND

Approximately half of EGFR-L858R and/or EGFR-ex-19del mutant non-smallcell lung cancer (NSLC) patients treated with small molecule EGFR kinaseinhibitors develop resistance associated with the EGF receptorEGFR-L858R-T790M or EGFR-ex19delT790M substitution. Indolocarbazolecompounds have been identified as potent and reversible inhibitors ofEGFR-L858R-T790M and EGFR-ex19delT790M that spare wild type EGFR [23].

EGFR mutations, including EGFR-exon 19 deletions and EGFR-L858R are themost frequent actionable genomic events in lung adenocarcinomas [24].

Tyrosinse kinase inhibitors (TKIs) such as osimertinib have beenapproved for and have been demonstrated to overcome EGFR-L858R-T790Mand/or EGFR-ex19delT790M resistance. However C797S mutation present inEGFR triple mutants (C797S/T90M/activation mutation), induces resistanceto osimertinib and other TKIs.

Currently, there are no effective therapeutic strategies to overcome theC797S/T90M/activation mutation (triple mutation)-mediated EGFR-TKIresistance. Brigatinib has been identified to be effective againsttriple mutation (C797S/T790M/activating-mutation) harbouring cells inpreclinical models [18].

Additional treatment modalities are needed.

SUMMARY

A high throughout mammalian two hybrid screening platform, MaMTH-DS,identified compounds described herein that are, for example inhibitorsof the kinase activities of Epidermal Growth Factor Receptor (EGFR)mutants. The compounds described herein may be useful as medicaments,for example for treating cancers with mutated EGFR such as drugresistant lung cancer comprising an EGFR C797 mutation, optionally aC797S mutation. Further, it is disclosed that the compounds showactivity alone and in combination with therapeutic anti-EGFR antibodies.

Accordingly, an aspect of the disclosure includes a method of inhibitingactivity of a mutant epidermal growth factor receptor (EGFR) in a cellcomprising contacting the cell with a compound selected from3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and astructurally related analog, salt or solvate thereof; midostaurin and asalt or solvate thereof; AZD7762 and a structurally related analog, saltor solvate thereof; and gilteritinib and a salt or solvate thereof; andmixtures thereof.

Another aspect includes a method of treating a subject afflicted with alung cancer having a mutant EGFR, optionally having a C797 mutation, themethod comprising administering to a subject in need thereof atherapeutically effective amount of a compound selected from3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and astructurally related analog, salt or solvate thereof; midostaurin and asalt or solvate thereof; AZD7762 and a structurally related analog, saltor solvate thereof; and gilteritinib and a salt or solvate thereof; andmixtures thereof.

Yet another aspect includes, a composition comprising at least twocompounds or a combination comprising at least two compounds selectedfrom:

a compound of formula I and/or structurally related analog, salt orsolvate thereof;

a compound of formula II, salt or solvate thereof;

a compound of formula III and a structurally related analog, salt orsolvate thereof;

a compound of formula IV, salt or solvate thereof; and/or

an anti-EGFR therapeutic antibody.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the application are given byway of illustration only, the scope of the claims should not be limitedby the embodiments set forth in the examples, but should be given thebroadest interpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1a-c Overview of key modifications employed in the MaMTH-DSplatform. (a) Schematic diagram of FLP-ln TREx compatible MaMTH-DS baitvector. (b) Methylene blue rinse test demonstrating enhanced adherenceof FLP-compatible HEK293 cells carrying randomly integrated macrophagescavenger receptor 1 (MSR1, bottom panel) vs FLP-compatible HEK293 WT(top panel). (c) Comparison of Firefly vs Gaussia princeps luciferaseactivity across EGFR WT and mutants in MaMTH assays performed in a384-well format. EGFR T790M=EGFR L858R/T790M, EGFR C797S=EGFRL858R/T790M/C797S.

FIG. 2a-e Effect of TKI therapeutics on the MaMTH-DS RTK signal. Panelsshow the effect of indicated compounds on MaMTH-DS activity in reportercells stably expressing RTK bait in the presence of transientlytransfected Shcl prey. (a) MET receptor with Crizotinib and Erlotinib.(b) FGFR4 receptor with BLU9931 and Erlotinib. (c) AXL receptor withForetinib and Erlotinib. (d) ALK receptor with Brigatinib and Erlotinib.(e) EGFR-WT and oncogenic mutants with Erlotinib, Rociletinib andOsimertinib.

FIG. 3a-d Effect of TKI therapeutics on MaMTH RTK baits. Left panelsshow the effect of the indicated compounds on the viability of reportercells expressing RTK bait. Right panels show the effect of indicatedcompounds on the expression of RTK bait in reporter cells. (a) METreceptor with Crizotinib. (b) FGFR4 receptor with BLU9931. (c) AXLreceptor with Foretinib. (d) ALK receptor with Brigatinib.

FIG. 4a-c Effect of TKI therapeutics on MaMTH reporter cells stablyexpressing EGFR WT and mutant baits. (a) Effect of reported TKItherapeutics on MaMTH EGFR bait reporter cell viability. (b) Effect ofreported TKI therapeutics on EGFR bait expression. (c) Effect ofreported TKI therapeutics on background MaMTH signal produced by EGFRbaits in the absence of prey.

FIG. 5 Effect of TKI molecules on MaMTH-DS signal in reporter cellsstably expressing MET, FGFR4, AXL or ALK receptor in the absence ofprey.

FIG. 6 Effect of therapeutic TKIs on endosomal trafficking of EGFR WTand mutants in MaMTH reporter lines. Left panels present total integralvesicular intensity of EGF (black) and EGF co-localized with EEA1 (red).Right panels show mean integral intensity of Y1068 phosphorylated EGFRendosomes (black) and mean over EEA1-positive endosomes (red) upon EGFstimulation and TKI treatment. Intensity distribution of pY1068 inun-stimulated (0 min) cells was subtracted from intensity distributionsat 20 and 30 min to correct for background signal. Os=Osimertinib.Ro=Rociletinib. Er=Erlotinib.

FIG. 7 Schematic representation of the MaMTH-DS platform workflow.

FIG. 8a-e MaMTH-DS screening of EGFR L858R/T790M/C797S in the presenceof Shcl. (a) Source and number of small molecules used in our pilotscreening library. Library included compounds from the MaybridgeHitFinder TM, Chembridge N1189-1 and Ontario Institute for CancerResearch (OICR) TKI collections. (b) Z′ values across all ten platesused in each round of screening. (c) Scatterplot of NPI vs BScore forall samples in screens. Horizontal red and green lines correspond to NPIvalues of 70% and −100%, respectively. Vertical red and green linescorrespond to BScore values of −3 and +3, respectively. All values inthe upper left quadrants were scored as hits. (d) Total hits and overlapfor screening Rounds 1 and 2. (e) Total mutant-specific anddose-responsive compounds identified upon retesting of shared hits fromRounds 1 and 2 of screening.

FIG. 9a-c MaMTH-DS screen of EGFR L858R/T790M/C797S in the presence ofShcl. (a) Comparison of raw reporter signal of EGFR L858R/T790M/C797SMaMTH bait cell line with and without transfected Shcl prey. (b) Sampledistributions of untransformed and Box-Cox power transformed data (usinga value of Lambda=0.71 and 0.87 for screening rounds 1 and 2,respectively). P-Values from Shapiro-Wilk's normality test of data areshown in inset. (c) Box and whisker plots showing distribution of samplevalues across plates for non-normalized, NPI-normalized andBScore-normalized data. Medians are indicated by thick black lines.Filled blue boxes encompass the 25th to 75th percentiles. Whiskersextend to the largest and smallest values not greater than 1.5 times theIQR. Outlying points beyond the whiskers are shown individually in red.

FIG. 10 MaMTH-DS hits from screen of EGFR L858R/T790M/C797S in thepresence of Shcl. Hit distributions across plates for Rounds 1 and 2 ofscreening are shown. The total number of hits per screen is labelledinset.

FIG. 11a-b MaMTH dose response analysis of top hits from MaMTH-DSscreens of EGFR L858R/T790M/C797S in the presence of Shcl. (a) Curvesfor the three candidates showing robust, dose-responsive and mutantspecific inhibition, selected for further analysis. (b) Curves for twocandidates not selected for further analysis.

FIG. 12 Effect of Chembridge 5213777 on viability of MaMTH-DS reportercells expressing EGFR WT or EGFR L858R/T790M/C797S.

FIG. 13a-d Functional analysis of Midostaurin against EGFR-C797S triplemutants. (a) Effect of Midostaurin on EGFR and downstream signallingmolecule expression and phosphorylation in Ba/F3 cells expressing EGFRL858R/T790M/C797S, EGFR ex19del/T790M/C797S or EGFR WT. (b) Effect ofMidostaurin on viability of Ba/F3 cells expressing EGFRL858R/T790M/C797S, EGFR ex19del/T790M/C797S or EGFR WT. (c,d) Enhancedeffect of Midostaurin in combination with 68 nM Cetuximab (CTX) or 140nM Panitumumab on Ba/F3 EGFR L858R/T790M/C797S and EGFRex19del/T790M/C797S cell viability. Brigatinib is included forcomparison. Statistical significance of differences in cell viabilitywere determined using the Student's t-test. ** indicates a p-Value lessthan 0.01.

FIG. 14a-d Functional analysis of 5213777 against EGFR C797S triplemutants. (a) Effects of 5213777 on EGFR and downstream signallingmolecule expression and phosphorylation in Ba/F3 cells expressing EGFRL858R/T790M/C797S, EGFR ex19del/T790M/C797S or EGFRWT. (b) Effects of5213777 on viability of Ba/F3 cells expressing EGFR L858R/T790M/C797S,EGFR ex19del/T790M/C797S or EGFR WT. (c,d) Enhanced effect of 5213777 incombination with 68 nM Cetuximab (CTX) or 140 nM Panitumumab on Ba/F3EGFR L858R/T790M/C797S and EGFR ex19del/T790M/C797S cell viability.Statistical significance of differences in cell viability weredetermined using the Student's t-test. ** indicates a p-Value less than0.01.

FIG. 15a-d Functional analysis of AZD7762 against EGFR C797S triplemutants. (a) Effects of AZD7762 on EGFR and downstream signallingmolecule expression and phosphorylation in Ba/F3 cells expressing EGFRL858R/T790M/C797S, EGFRex19del/T790M/C797S or EGFRWT. (b) Effects ofAZD7762 on viability of Ba/F3 cells expressing EGFR L858R/T790M/C797S,EGFR ex19del/T790M/C797S or EGFR WT. (c,d) Enhanced effect of AZD7762 incombination with 68 nM Cetuximab (CTX) or 140 nM Panitumumab on Ba/F3EGFR L858R/T790M/C797S and EGFR ex19del/T790M/C797S cell viability.Statistical significance of differences in cell viability weredetermined using the Student's t-test. * and ** indicate a p-Value lessthan 0.05 and 0.01 respectively.

FIG. 16a-b Effect of Osimertinib on Ba/F3 cell viability in the presenceof anti-EGFR therapeutic antibodies. (a,b) Osimertinib in combinationwith 68 nM Cetuximab (CTX) or 140 nM Panitumumab does not cause enhancedreduction of Ba/F3 EGFR L858R/T790M/C797S or EGFR ex19del/T790M/C797Scell viability, consistent with the inability of this compound to targetC797S mutants.

FIG. 17a-d In vitro kinase assays of identified compounds against EGFRWT and oncogenic triple mutants. (a) Midostaurin. (b) Brigatinib. (c)AZD7762. (d) Chembridge 5213777.

FIG. 18a-b Effect of Midostaurin on EGFR kinase activity and signalling.(a) Midostaurin inhibits the kinase activity of EGFR double and triplemutants. (b) Midostaurin inhibits EGFR activation and downstreamsignaling in PC9 EGFR ex19del/T790M/C797S cells.

FIG. 19a-b Effect of Midostaurin on PC9 cells expressing EGFR C979Striple mutants. (a) Midostaurin, but not Osimertinib, activates caspase3 and 7 activity in PC9 EGFR ex19del/T790M/C797S cells but not CBFEcells. (b) Midostaurin, but not Osimertinib, reduces the viability ofPC9 EGFR ex19del/T790M/C797S organoids.

FIG. 20a-c Effect of 5213777 on MaMTH-DS signal, EGFR kinase activityand downstream signalling. (a) 521377 inhibits the interaction betweenShc1 and EGFR L858R/T790M/C797S but not EGFR WT, MET, FGFR4, AXL or ALK.(b) 521377, but not Osimertinib, inhibits the interaction between EGFRL858R/T790M/C797S and Shc1, Crkll and Hsp90. (c) 521377 does not inhibitthe kinase activity of EGFR L858R/T790M/C797S or EGFRex19del/T790M/C797S in in vitro kinase assays.

FIG. 21a-d Effect of 521377 on PC9 cells expressing EGFR C979S triplemutants. (a) 521377 inhibits cell viability of PC9 EGFR ex19del, PC9EGFR ex19del/T790M and PC9 EGFR ex19del/T790M/C797S cells but not CFBEEGFR WT cells. (b) 521377, but not Osimertinib, activates caspase 3 and7 activity in PC9 EGFR ex19del/T790M/C797S cells but not CBFE cells. (c)521377 reduces the viability of PC9 EGFR ex19del/T790M/C797S organoids,(d) 521377 inhibits EGFR activation and downstream signaling in PC9 EGFRex19del/T790M/C797S cells.

FIG. 22a-c Effect of Gilteritinib on MaMTH-DS signal, EGFR kinaseactivity and downstream signalling. (a) Gilteritinib preferentiallyinhibits the interaction between Shc1 and EGFR L858R/T790M or EGFRL858R/T790M/C797S but not EGFR WT. (b) Gilteritinib inhibits the kinaseactivity of EGFR L858R/T790M/C797S, EGFR ex19del/T790M and EGFRex19del/T790M/C797S in in vitro kinase assays. (c) Gilteritinib inhibitsEGFR activation and downstream signaling in PC9 EGFR ex19del/T790M/C797Scells.

FIG. 23a-b Functional analysis of Gilteritinib against EGFR C797S triplemutants. (a) Gilteritinib but not Osimertinib reduces the viability ofPC9 EGFR ex19del/T790M/C797S organoids. (b) Enhanced effects ofGilteritinib (left panel) or Midostaurin (right panel) in combinationwith 10 microgram/ml Panitumumab on PC9 EGFR ex19del/T790M/C797S cellviability.

FIG. 24 is a series of graphs and tables demonstrating effect ofexemplary compounds of the disclosure.

FIG. 25a-b is a series of graphs and tables demonstrating effect ofexemplary mixtures of the disclosure.

DETAILED DESCRIPTION A. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the application herein described for which they aresuitable as would be understood by a person skilled in the art.

As used in this application, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.For example, an embodiment including “a compound” should be understoodto present certain aspects with one compound, or two or more additionalcompounds.

In embodiments comprising an “additional” or “second” component, such asan additional or second compound, the second component as used herein ischemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. The term “consisting” and its derivatives, as used herein,are intended to be closed terms that specify the presence of the statedfeatures, elements, components, groups, integers, and/or steps, butexclude the presence of other unstated features, elements, components,groups, integers and/or steps. The term “consisting essentially of”, asused herein, is intended to specify the presence of the stated features,elements, components, groups, integers, and/or steps as well as thosethat do not materially affect the basic and novel characteristic(s) offeatures, elements, components, groups, integers, and/or steps.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.”

The term “compound(s) of the disclosure” or “compound(s) of the presentdisclosure” and the like as used herein means compounds of formula I andstructurally related analogs, or a pharmaceutically acceptable salt orsolvate thereof, compounds of formula II, or a pharmaceuticallyacceptable salt or solvate thereof, compounds of formula III andstructurally related analogs, or a pharmaceutically acceptable salt orsolvate thereof, and compounds of formula IV, or a pharmaceuticallyacceptable salt or solvate thereof, and includes tautomers,regioisomers, geometric isomers, and where applicable, stereoisomers,including optical isomers (racemic mixtures, enantiomers, orenantiomerically enriched mixtures) and other stereoisomers(diastereomers) thereof.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects, in particular humans.

The term “pharmaceutically acceptable salt” means an acid addition saltor a base addition salt which is suitable for, or compatible with, thetreatment of subjects.

An acid addition salt which is suitable for, or compatible with, thetreatment of subjects as used herein means any non-toxic organic orinorganic salt of any basic compound. Basic compounds that form an acidaddition salt include, for example, compounds comprising an amine group.Illustrative inorganic acids which form suitable salts includehydrochloric, hydrotrifluoroacetic, hydrobromic, sulfuric and phosphoricacids, as well as metal salts such as sodium monohydrogen orthophosphateand potassium hydrogen sulfate. Illustrative organic acids that formsuitable salts include mono-, di-, and tricarboxylic acids such asglycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic,tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic andsalicylic acids, as well as sulfonic acids such as p-toluene sulfonicand methanesulfonic acids. Either the mono- or di-acid salts can beformed, and such salts may exist in either a hydrated, solvated orsubstantially anhydrous form. In general, acid addition salts are moresoluble in water and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of the appropriate salt will be known to oneskilled in the art. In an embodiment, the acid addition salt is ahydrochloride or hydrotrifluoroacetic acid salt.

A base addition salt which is suitable for, or compatible with, thetreatment of subjects as used herein means any non-toxic organic orinorganic base addition salt of any acidic compound. Acidic compoundsthat form a base addition salt include, for example, compoundscomprising a carboxylic acid group. Illustrative inorganic bases whichform suitable salts include lithium, sodium, potassium, calcium,magnesium or barium hydroxide. Illustrative organic bases which formsuitable salts include aliphatic, alicyclic or aromatic organic aminessuch as methylamine, trimethylamine and picoline, alkylammonias orammonia. The selection of the appropriate salt will be known to a personskilled in the art.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The term “solvate” as used herein means a compound or itspharmaceutically acceptable salt, wherein molecules of a suitablesolvent are incorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”. The formation ofsolvates will vary depending on the compound and the solvate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions.

The term “dosage form” as used herein refers to the physical form of adose for example comprising a desired compound of the disclosure, andincludes without limitation injectable dosage forms, including, forexample, sterile solutions and sterile powders for reconstitution, andthe like, that are suitably formulated for injection, liquid and soliddosage forms including, for example tablets, including enteric coatedtablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets,troches, elixirs, suspensions, syrups, wafers, resuspendable powders,liquids and solutions.

The term “diluent” as used herein refers to a pharmaceuticallyacceptable carrier which does not inhibit a physiological activity orproperty of an active compound to be administered and does not irritatethe subject and does not abrogate the biological activity and propertiesof the administered compound. Diluents include any and all solvents,dispersion media, coatings, surfactants, antioxidants, preservativesalts, preservatives, binders, excipients, disintegration agents,lubricants, such like materials and combinations thereof, as would beknown to one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with drugresistant lung cancer can be treated to prevent progression, oralternatively a subject in remission can be treated with a compound orcomposition described herein to prevent recurrence. Treatment methodscomprise administering to a subject a therapeutically effective amountof one or more of the compounds of the disclosure and optionally,consists of a single administration, or alternatively comprises a seriesof administrations. For example, the compounds are administered to thesubject in an amount and for a duration sufficient to treat the patient.

The term “drug resistant lung cancer” means a lung cancer that has atleast one drug resistance mutation, optionally two drug resistancemutations, such as EGFR triple mutants comprising C797S/T790M mutationsand/or a lung cancer that has progressed in a subject on at least 1 EGFRinhibitor.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve the desired result. Effective amounts mayvary according to factors such as the disease state, age, sex and/orweight of the subject. The amount of a given compound that willcorrespond to such an amount will vary depending upon various factors,such as the given drug or compound, the pharmaceutical formulation, theroute of administration, the type of condition, disease or disorder, theidentity of the subject being treated, and the like, but cannevertheless be routinely determined by one skilled in the art.

The term “mixture” as used herein refers to a composition comprising twoor more compounds, salts or solvates. The term combination includes twoor more compounds or two or more compositions each comprising one ormore compounds, salts or solvates thereof including mixtures of any ofthe foregoing.

The term “triple mutant EGFR” as used herein means EGFR comprising anactivating mutation such as ex19del or L858R and the resistancemutations at T790 (e.g. T790M) and C797 (e.g. C797S) and in particularto one of EGFR-ex19del/T790M/C797S and EGFR-L858R/T790M/C797S mutants.

The term “administered” as used herein means administration of atherapeutically effective dose of a compound or composition of theapplication to a cell either in cell culture or in a patient (i.e.subject) by any means of administration suitable.

The term “in combination” or “combination therapy” as used herein meansthat at least two compounds or compositions are administered to thepatient as part of a treatment regimen, administered for examplecontemporaneously, sequentially and/or in alternating fashion,optionally such that effective amounts or concentrations of each of thetwo or more compounds may be found in the patient at a given point intime. Although compounds according to the present disclosure may beco-administered to a patient at the same time, the term embraces bothadministration of two or more compounds of the disclosure at the sametime or at different times and encompasses where the effectiveconcentrations of all coadministered compounds or compositions are foundin the subject at a given time.

The term “non-small cell lung cancer” as used herein includesadenocarcinomas, squamous cell carcinomas, large cell carcinomas,adenosquamous carcinoma and sarcomatoid carcinoma.

The term “mutant epidermal growth factor receptor (EGFR)” as used hereinmeans an EGFR having at least one activating mutation associated withdisease. The mutant EGFR can also comprise additional mutations,including drug resistance mutations such as T790M and/or C797S.

The term “3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one andstructurally related analogs thereof” as used herein refers to thecompound of formula (I):

and structurally related analogs thereof including benzoxazolyl and/orchromenone containing compounds, substituted versions thereof or a saltor solvate of any of the foregoing as well as mixtures thereof,hereinafter: “Compound of formula I and structurally related analogsthereof”. Reference to a compound of formula I and/or a structurallyrelated analog thereof thereby refers to one of a compound of formula I,a structurally related analog thereof, a salt of any of the foregoingand/or a mixture of any of the foregoing. The structurally relatedanalogs contemplated include or are molecules sharing the same backboneand which can inhibit mutant EGFR, preferably triple mutant EGFRcomprising mutation of C797, interaction with Shcl. This compound isalternatively referred to by as3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, Chembridge5213777, Compound 5213777, and 5213777. Also, structural analogs includehalogen derivatives on the benzoxazolyl moiety of compound3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, for examplechlorinated derivatives thereof such as3-(5-chloro-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, alsoreferred as to CAS #35773-43-4, MolPort-002-557-047, or Disperse Yellow232, which is available from a number of vendors including AK ScientificInc.

Derivatives of chromen-2-one are described in US 20060122387 and areincorporated herein by reference, including the ones mentioned below.

 1

7-Methoxy-3-(2-methyl- thiazol-4-yl)-chro-men- 2-one  2

[4-(6-Bromo-2-oxo-2H- chromen-3-yl)-thia-zol- 2-yl]-acetonitrile  3

7-Ethoxy-3-(2-methyl- thiazol-4-yl)-chro-men- 2-one  4

Acetic acid 6-hexyl-3- (4-methyl-thia-zol-2- yl)-2-oxo-2H-chromen- 7-ylester  5

3-(2-[4-(Chromen-2- one-3-yl)tiazol-2-yl]- thia-zol-4-yl)-chromen- 2-one 6

7-Methoxy-3-(2-methyl- thiazol-4-yl)-chro-men- 2-one  7

Acetic acid 2-oxo-3-(4- phenyl-thia-zol-2-yl)- 2H-chromen-7-yl ester  8

7-Hydroxy-3-(2-phenyl- thiazol-4-yl)-chro-men- 2-one  9

6-Methoxy-3-[4-(4- methoxy-phenyl)-thia- zol-2-yl]-chromen-2- one 10

7-Hydroxy-3-[4-(4- methoxy-phenyl)-thia- zol-2-yl]-chromen-2- one 11

7-Diethylamino-3-[2- (4-dimethylamino- phenyl)-thiazol-4-yl]-chromen-2-one 12

Acetic acid 6-hexyl-3- (2-methyl-thiazol-4- yl)-2-oxo-2H-chromen- 7-ylester 13

3-[4-(4-Chloro-phenyl)- thiazol-2-yl]-6-hex-yl- 7-hydroxy-chromen-2- one14

6-Hexyl-7-hydroxy-3- [4-(4-phenoxy-phe-nyl)- thiazol-2-yl]-chromen-2-one 15

7-Hydroxy-3-[4-(4- phenoxy-phenyl)-thia- zol-2-yl]-chromen-2- one 16

6-Hexyl-7-hydroxy-3- [4-(4-methoxy-phe- nyl)-thiazol-2-yl]-chromen-2-one 17

7-Diethylamino-3-(4- methyl-thiazol-2-yl)- chro-men-2-one 18

3-[4-(4-Bromo-phenyl)- thiazol-2-yl]-6-hex-yl- 7-hydroxy-chromen-2- one19

6-Hexyl-7-hydroxy-3- (2-phenyl-thia-zol-4- yl)-chromen-2-one 20

6-Hexyl-7-hydroxy-3- (4-phenyl-thiazol-2-yl)- chromen-2-one 21

7-Hydroxy-3-(4-phenyl- thiazol-2-yl)-chro-men- 2-one 22

6-Hexyl-7-hydroxy-3- (4-methyl-thiazol-2-yl)- chromen-2-one 23

Acetic acid 3-[4-(2,5- dimethyl-phe-nyl)-5- ethyl-thiazol-2-yl]-2-oxo-2H-chro-men-7-yl ester 24

7-Hydroxy-3-(5-methyl- 4-phenyl-thiazol-2-yl)- chromen-2-one 25

3-[4-(4-Chloro-4- phenyl)-5-methyl- thiazol-2-yl]-7- hydroxy-chromen-2-one 26

7-Hydroxy-3-(5-methyl- 4-p-tolyl-thiazol-2-yl)- chromen-2-one 27

Acetic acid 3-(4,5- dihydro-naphtho[1,2- d]thia-zol-2-yl)-2-oxo-2H-chromen-7-yl ester 28

Acetic acid 2-oxo-3-[4- (5,6,7,8-tetra-hydro- naphthalen-2-yl)-thiazol-2-yl]-2H-chro-men-7-yl ester 29

6-Bromo-3-[4-(4- ethoxy-phenyl)-thia- zol-2-yl]-chromen- 2-one 30

3-[4-(4-Ethyl-phenyl)- 5-methyl-thiazol-2-yl]- 7-hydroxy-chromen-2- one31

3-[4-(4-Chloro-phenyl)- thiazol-2-yl]-6-meth- oxy-chromen-2-one 32

3-[2-(3,4-Dimethoxy- phenyl)-thiazol-4-yl]- chro-men-2-one 33

3-[4-(4-Bromo-phenyl)- 5-ethyl-thiazol-2-yl]-7- hydroxy-chromen-2-one 34

Acetic acid 3-[4-(4- bromo-phenyl)-5-eth- yl-thiazol-2-yl]-2-oxo-2H-chro-men-7-yl ester 35

3-(4,5-Dihydro- naphtho[1,2-d]- thiazol-2-yl)-7-hy- droxy-chromen- 2-one36

7-Diethylamino-3-(4- phenyl-thiazol-2-yl)- chro-men-2-one 37

Acetic acid 3-[5-ethyl- 4-(4-ethyl-phe-nyl)- thiazol-2-yl]-2-oxo-2H-chro-men-7-yl ester 38

Acetic acid 3-[4-(4- chloro-phenyl)-5-eth- yl-thiazol-2-yl]-2-oxo-2H-chro-men-7-yl ester 39

3-[4-(3,4-Dichloro- phenyl)-5-methyl- thiazol-2-yl]-7- hydroxy-chromen-2-one 40

3-[4-(4-Chloro-phenyl)- 5-ethyl-thia-zol-2-yl]-7- hydroxy-chromen-2-one41

3-(5-Ethyl-4-p-tolyl- thiazol-2-yl)-7-hy- droxy-chromen-2-one 42

7-Diethylamino-3-(2- phenyl-thiazol-4-yl)- chro-men-2-one 43

3-[4-(2,5-Dimethyl- phenyl)-5-ethyl- thiazol-2-yl]-7- hydroxy-chromen-2-one 44

Acetic acid 3-[4-(2,4- dimethyl-phenyl)-5- ethyl-thiazol-2-yl]-2-oxo-2H-chro-men-7- yl ester 45

3-[2-(4-Hydroxy- phenyl)-thiazol-4-yl]- chro-men-2-one 46

3-(5-Ethyl-4-phenyl- thiazol-2-yl)-7-hy- droxy-chromen-2-one 47

3-[2-(2,4-Dimethyl- phenyl)-thiazol-4-yl]- 7-hy-droxy-chromen- 2-one 48

3-[4-(3-Bromo-phenyl)- thiazol-2-yl]-8-meth- oxy-chromen-2-one 49

7-Hydroxy-3-[4-(3- methoxy-phenyl)-thia- zol-2-yl]-chromen-2- one 50

3-Benzothiazol-2-yl-7- hydroxy-chro-men-2- one 51

3-(5-Chloro-1H- benzoimidazol-2-yl)- 8-meth-oxy-chromen- 2-one 52

2-(5-Phenyl-[1,3,4]- oxadiazol-2-yl)-ben- zo[f]chromen-3-one 53

2-Benzooxazol-2-yl- benzo[f]chromen-3- one 54

3-[5-(3-Fluoro-phenyl)- [1,3,4]oxa-diazol-2-yl]- 8-methoxy-chromen-2-one 55

3-Benzo[d]imidazo[2,1- b]thiazol-2-yl-chro-men- 2-one 56

3-(7-Methoxy-benzo- [d]imidazo[2,1-b]thia- zol-2-yl)-chromen-2- one 57

6-Chloro-3-(7-fluoro- benzo[d]imi-dazo- [2,1-b]thiazol-2-yl)-chromen-2-one 58

2-(4-Thiosemicar- bazidomethyl-1-phe- nyl-1H-pyrazol-3-yl)-benzo[f]chromen-3- one 59

(3-Benzooxazol-2-yl- 2-oxo-2H-chro-men-7- yloxy)-acetic acid ethyl ester60

3-Benzo[d]imidazo[2,1- b]thiazol-2-yl-6-hex-yl- 7-hydroxy-chromen-2- one61

6,8-Dichloro-3-(5-p- tolylamino-[1,3,4]thia- diazol-2-yl)-chromen- 2-one62

3-[5-(3-Chloro-phenyl)- [1,3,4]oxa-diazol-2-yl]- 6-hexyl-7-hydroxy-chro-men-2-one 63

6-Hexyl-7-hydroxy-3- [5-(3-methoxy-phe-nyl)- [1,3,4]oxadiazol-2-yl]-chro-men-2-one 64

6-Hexyl-7-hydroxy-3- (7-methyl-imidazo- [1,2-a]pyridin-2-yl)-chromen-2-one 65

6-Methoxy-3-[2-(4- methoxy-phenyl- amino)-thiazol-5-yl]- chromen-2-one66

6-Hexyl-7-hydroxy-3- [5-(3,4,5-trimeth-oxy- phenyl)-[1,3,4]oxa-diazol-2-yl]-chro-men- 2-one

3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one is compound5213777 of the Chembridge Diverset library (e.g. N1189-1) of compoundsand can be purchased from ChemBridge Online Chemical Store:https://www.hit2lead.com/.

“Midostaurin” as used herein refers toN-[(9S,10R,11R,13R)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiazonin-11-yl]-N-methylbenzamideof the formula (II):

or a salt or solvate thereof, hereinafter: “Compound of formula II ormidostaurin”.

Midostaurin is also known as 4-N-benzoyl staurosporine,Benzoylstaurosporine, CGP 41251, N-benzoyl-staurosporine, PKC412,PKC412A, and Rydapt™ and is a derivative of the naturally occurringalkaloid staurosporine. It has been specifically described in theEuropean patent No. 0 296 110 published on Dec. 21, 1988, as well as inU.S. Pat. No. 5,093,330 published on Mar. 3, 1992, and Japanese PatentNo. 2 708 047. Midostaurin described in these documents are incorporatedinto the present application by reference. Midostaurin is a compound inthe OICR TKI library with identifier OICR0000317A01. Midostaurin isavailable from a number of vendors including Sigma-Aldrich. Midostaurinand its manufacturing process have been described.

The term “AZD7762” as used herein refers3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamideof the formula (III):

or a salt or solvate thereof, hereinafter as well as structurallyrelated compounds that inhibit triple mutant EGFRs: hereinafter compoundof formula III and structurally related analogs. The structurallyrelated analogs contemplated include or are other pyrazolidinedionederivatives, for example as described in WO2005002574 hereinincorporated by reference, sharing the same backbone as AZD7762 andwhich can inhibit mutant EGFR, preferably triple mutant EGFR comprisingmutation of C797, interaction with Shcl. In an embodiment the salt isthe hydrochloride. “Compound of formula III or AZD7762”.

AZD7762 is a compound in the OICR TKI library with identifierOICR0001145B01, Pubchem CID 11152667 and a formula C₁₇H₁₉FN₄O₂S. AZD7762is available for purchase from a number of vendors includingSigma-Aldrich.

“Gilteritinib” as used herein refers to6-ethyl-3-[3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]anilino]-5-(oxan-4-ylamino)pyrazine-2-carboxamideof the formula (IV):

or a salt or solvate thereof, hereinafter: “Compound of formula IV orgilteritinib”.

Gilteritinib, also known as ASP2215, is a receptor tyrosine kinaseinhibitor of FLT3 and AXL with inhibitory activity against FLT3 internaltandem duplication (ITD) as well as tyrosine kinase domain (TKD), twocommon types of FLT3 mutations that are seen in up to one third ofpatients with acute myeloid leukemia (AML). Gilteritinib, as well asstructurally related analog compounds, have been described inPCT/JP2010/057751 published on Nov. 11, 2010, as well as in U.S. Pat.No. 8,969,336, and U.S. Pat. No. 9,487,491, which are incorporated intothe present application by reference. In a particular embodiment, thepreferred salt is fumarate. Gilteritinib is available from a number ofvendors including AK Scientific.

Cetuximab, also known as Erbitux™, is an anti-EGFR therapeutic antibodycapable of inhibiting the growth of human tumor cells expressing humanEGFR. Cetuximab is described in U.S. Pat. No. 6,217,866, which isincorporated into the present application by reference. Cetuximab isavailable from a number of vendors including BioRad.

Panitumumab, also known as Vectibix™, is a fully human monoclonalantibody against human EGFR. Panitumumab is described in U.S. Pat. No.6,235,883, which is incorporated into the present application byreference. Panitumumab is available from a number of vendors includingBioRad.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

B. Methods

Described herein are compounds, compositions and methods for inhibitingEGFR mutant complex formation and signalling that may be useful for thetreatment for treating EGFR-mutant lung cancers.

Accordingly, an aspect of the disclosure provides a method of inhibitingactivity of a mutant epidermal growth factor receptor (EGFR) in a cellcomprising contacting the cell with a compound selected from3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and astructurally related analog thereof; midostaurin or a salt or solvatethereof; AZD7762 and a structurally related analog thereof; gilteritinibor a salt or solvate thereof; and mixtures thereof.

As demonstrated in the Examples mutant EGFR comprising a C797 mutation,such as the C797S mutation that arises in lung cancers that have becomeresistant to TKIs such as osimertinib that are effective for inhibitingactivity of and for treating cancers comprising EGFR double mutants suchas L858R-T790M and ex19Del-T790M, is shown to be sensitive to compounds(“compounds of the disclosure”) and combination therapies describedherein.

It is demonstrated for example that the compounds of the disclosureinhibit mutant EGFR endosomal trafficking, mutant EGFR phosphorylation,mutant EGFR expression, mutant EGFR signaling, mutant EGFR proliferationand/or Shc1 complex formation.

Accordingly in one embodiment, the activity comprises inhibitinginteraction of the mutant EGFR with Shc1 thereby inhibiting complexformation. In other embodiments, the activity is selected from mutantEGFR endosomal trafficking, mutant EGFR phosphorylation, mutant EGFRexpression, mutant EGFR kinase activity, mutant EGFR signaling and/ormutant EGFR proliferation.

The cell can be any cell comprising mutant EGFR such as an activatingmutation and/or a drug resistance mutation, optionally combined, forexample combined with a drug resistance mutation C797 mutation. In anembodiment the cell is a lung cancer cell optionally non-small cell lungcancer cell. The cell can be in vitro or in vivo.

EGFR is often mutated in lung cancer and drugs have been developed totarget mutant EGFR (e.g. mutations in the TK domain). Lung cancers candevelop resistance and drug resistant lung cancers where C797 is mutatedhave been identified. It is demonstrated that compounds of thedisclosure as identified in the Examples, reproducibly inhibit in a dosedependent manner cells expressing C797 mutated EGFR.

Accordingly also provided is a method of treating a subject afflictedwith a lung cancer, optionally having a mutant EGFR comprising a C797mutation, the method comprising administering to a subject in needthereof a therapeutically effective amount of a compound selected from3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and astructurally related analog thereof; midostaurin or a salt or solvatethereof; AZD7762 and a structurally related analog thereof; gilteritinibor a salt or solvate thereof; and mixtures thereof.

Several TKIs such as osimertinib have been approved and demonstrated toovercome EGFR-T790M resistance. However, drug resistant triple mutantlung cancers have developed. In particular, C797S mutation present inEGFR triple mutants (C797S/T790M/activation mutation), inducesresistance to osimertinib and other TKIs.

In an embodiment, the lung cancer is a drug-resistant lung cancer,optionally a drug-resistant cancer associated with EGFR C797 mutation,optionally wherein the C797 mutation is C797S.

In an embodiment, the drug resistant lung cancer comprises EGFRmutations L858R-T790M-C797S or ex19Del-T790M-0797S.

The ex19del refers to small, in frame deletions occurring in exon19 ofEGFR (which encodes part of the kinase domain). They primarily occurbetween codons 746 to 759. This is one of the most prominent EGFRmutations in lung cancer, occurring in about 40% of all EGFR-positiveNSCLC patients.

In an embodiment, the lung cancer is a NSCLC. The NSCLC can be anadenocarcinoma, squamous cell carcinoma, large cell carcinoma,adenosquamous carcinoma or a sarcomatoid carcinoma.

In one embodiment, the lung cancer is a locally advanced or metastaticnon-small cell lung cancer that has failed at least one priorchemotherapy regimen.

In an embodiment, the subject is administered a mixture of thecompounds. For example the mixture can be3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one andmidostaurin, midostaurin and AZD7762,3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and AZD7762,gilteritinib and3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, gilteritiniband midostaurin, gilteritinib and AZD7762 or structurally relatedanalogs of any of the foregoing combined with3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, midostaurin,gilteritinib or AZD7762 or any combination thereof.

The compounds or compositions can also be administered in combination,for example where the compounds are administered in separatecompositions, optionally contemporaneously, sequentially as part of atreatment regimen, such as a chemotherapy treatment regimen.

It is also demonstrated that increased (e.g. synergistic) activity isseen when the compound of the disclosure is administered with ananti-EGF antibody. As demonstrated in the examples, the toxicity of3-(1,3-benzoxazol-2-yl-7-(diethylamino)-2H-chromen-2-one, midostaurin,or AZD7762 towards EGFR triple mutant expressing cells is increased whenadministered with an anti-EGFR therapeutic antibody, for examplecetuximab or panitumumab.

A demonstrated in the examples, gilteritinib administered with ananti-EGFR therapeutic antibody, Panitumumab, produces an increasedtoxicity to triple mutant expressing cells (see FIG. 23b ). It isexpected that combining Gilteritinb with Cetuximab would also have anincreased toxicity.

Accordingly, in some embodiments, the compound or mixture isadministered as a combination therapy in combination with an anti-EGFtherapeutic antibody.

In particular embodiments, wherein the subject is administered acompound of formula I, formula II formula III, or formula IV, orstructural analogs of formula I or III or a mixture thereof, the subjectis also administered an anti-EGFR therapeutic antibody (e.g. aneutralizing anti-EGFR monoclonal antibody).

In an embodiment, the anti-EGFR therapeutic antibody is selected fromCetuximab or an antigen binding fragment thereof and Panitumumab or anantigen binding fragment thereof. Mimetics targeting the same region orepitope of EGFR as Cetuximab or Panitumumab can also be used.

The treatments can also be combined with one or more other treatmentsfor lung cancer.

The treatment for lung cancer can for example be surgery, chemotherapy,adjuvant therapy, radiation therapy, other targeted therapy or acombination thereof.

For example, the compounds, compositions and combinations of thedisclosure can be combined with a chemotherapy selected fromcarboplatin, cisplatin, docetaxel, gemcitabine, Nab-paclitaxel,premetresed and vinorelbine.

In some embodiments, the subject is confirmed to have one or more EGFRmutations. For example a sample (e.g. a tissue sample, a lung cancerbiopsy or a liquid biopsy such as a blood sample or plasma sample fordetecting tumour derived DNA and/or circulating tumour cells orcirculating exosomes) from the subject is tested for the presence of themutation. Suitable methods for obtaining tissue samples include tissuebiopsy, endobronchial biopsy, transbronchial biopsy, brushing cytology,washing cytology, fine needle aspiration cytology, fluid cytology, orbone biopsy. Testing for EGFR mutations can be done by any suitableanalytic technique, including quantitative real-time polymerase chainreaction (PCR), allele-specific PCR, or nucleic acid sequencing.Suitable tests include therascreen® EGFR RGQ PCR kit (Qiagen), cobas®EGFR Mutation Test v2 (Roche), FoundationOne CDx™ (Foundation Medicine),Oncomine™ Dx Target Test (Thermo Fisher Scientific), Guardant360™(Guardant Health), GeneStrat (Biodesix), OncoBEAM™ (Sysmex Inostics),ExoDx® Lung(T790M) (Exosome Diagnostics), and Biocept liquid biopsy. Ifthe mutation is present, the subject is administered a treatment asdescribed herein.

Uses of the compounds for treating lung cancer, preferably drugresistant mutant EGFR lung cancers, are also provided.

C. Compositions and Combinations

A further aspect includes a composition or combination comprising atleast two compounds selected from:

3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and/orstructurally related analog thereof;

midostaurin or a salt or solvate thereof;

AZD7762, and/or structurally related analog thereof;

gilteritinib or a salt or solvate thereof; and

an anti-EGFR therapeutic antibody.

For example, the composition can comprise or the combination can be3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one andmidostaurin; benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one andAZD7762; 3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one andgilteritinib; midostaurin and gilteritinib; AZD7762 and gilteritinib; ormidostaurin and AZD7762. In other embodiments, the composition orcombination comprises a structural analog of a compound of formula I orIII. In other embodiments, the combination is a compound of formula I ora structurally related analog and an anti-EGFR therapeutic antibody; acompound of formula II and an anti-EGFR therapeutic antibody; a compoundof formula III or a structurally related analog thereof and an anti EGFRtherapeutic antibody; or a compound of formula IV and an anti-EGFRtherapeutic antibody.

The combination can be used for combination treatments. The componentscan be packaged separately or together, for use in conjunction orsequentially.

The composition can comprise in addition one or more pharmaceuticallyacceptable carriers or diluents.

In an embodiment the composition is a pharmaceutical composition.

The pharmaceutical composition can comprise two or more compounds asdescribed above and a pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical composition is in a dosage formselected from a solid dosage form and a liquid dosage form.

In an embodiment, the pharmaceutical composition is administered byparenteral, intravenous, subcutaneous, intracardial, intramuscular, ororal administration.

In an embodiment, the pharmaceutical composition is an injectable dosageform.

In an embodiment, the injectable liquid is an injectable liquid depotsuitable, for example suitable for subcutaneous administration.

The formulations can be administered orally, topically, parenterally, byinhalation or spray, or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term “parenteral” as used herein includespercutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike. One or more compounds of the disclosure can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions of the disclosure can be ina form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed. Formulations fororal use can also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the compounds of the disclosure in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present. Pharmaceutical compositions of thedisclosure can also be in the form of oil-in-water emulsions. The oilyphase can be a vegetable oil or a mineral oil or mixtures of these.Suitable emulsifying agents can be naturally-occurring gums, for examplegum acacia or gum tragacanth, naturally-occurring phosphatides, forexample soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol, anhydrides, for example sorbitan monooleate,and condensation products of the said partial esters with ethyleneoxide, for example polyoxyethylene sorbitan monooleate. The emulsionscan also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art.

For example, a suitable dose of compound of formula (I) is in the rangeof about 0.1 to about 250 mg per kilogram body weight of the subject perday. For example, a suitable dose of compound of formula (II) is in therange of about 1 to 1000 mg, preferably of about 5 to about 500 mg, morepreferably from 10 to 100 mg per kilogram body weight of the subject perday. Even more preferably, a suitable dose of compound of formula (II)is 50 mg orally twice daily with food, or 100 orally twice daily withfood. For example, a suitable dose of compound of formula (IV), for oraladministration, is in the range of about 0.001 to 100 mg/kg, preferably0.005 to 30 mg/kg, and more preferably 0.01 to 10 mg/kg body weight, andeven more preferably in a dose from 20 to 450 mg, given as a single doseor in 2 to 4 divided doses. For intravenous administration, a suitabledose of compound of formula (IV) is in the range of about 0.0001 to 10mg/kg body weight, given in one or several doses per day. Fortransmucosal formulations, a suitable dose of compound of formula (IV)is in the range of about 0.001 to 100 mg/kg body weight, given in one orseveral doses per day. Selection of the lower range of concentration ordose for a given compound and/or analogue or combination thereof can bedetermined for example based upon, e.g., the EC₅₀ or ED₅₀ of thecomposition in established biological assays.

Suitable pharmaceutically acceptable carriers include essentiallychemically inert and nontoxic compositions that do not interfere withthe effectiveness of the biological activity of the pharmaceuticalcomposition. Examples of suitable pharmaceutical carriers include, butare not limited to, water, saline solutions, ethanol, polyethyleneglycol, propylene glycol, glycerin, castor oil, corn oil, gelatins,liposomes, natural polymers, synthetic polymers, polymeric blends,titanium dioxide, vitamins, coloring or pigment agents, hydroxypropylmethylcellulose, and the like.

In an embodiment, wherein the combination comprises a compound offormula I, formula II, formula III, and/or formula IV, the combinationfurther comprises an anti-EGFR therapeutic antibody.

In an embodiment, the anti-EGFR therapeutic antibody is selected fromCetuximab and an antigen binding fragment thereof, and Panitumumab andan antigen binding fragment thereof.

Each compound in the kit or combination can be packaged separately in aseparate housing such as a sterile vial or together in a single housingsuch as a single sterile vial.

The composition can be a pharmaceutical composition, optionallycomprising one of more pharmaceutically acceptable excipients ordiluents.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

To develop this new platform, which we have called MaMTH-DS (for MaMTH‘Drug Screening’), we introduced a number of significant modificationsto our traditional MaMTH system. The first modification was a transitionfrom transiently transfected to stably expressed integral membrane baitproteins, a necessary step to minimize variability/noise and allow forsensitive detection of small-molecule activity in a large-scalemulti-well format. To improve ease of stable generation, we developedreporter cell lines and a MaMTH-DS bait vector construct using theFlp-lN TREx system (Thermo Fisher), a Flp recombinase-based method whichallows for rapid generation of isogenic stables in as little as two tothree weeks. MaMTH-DS bait vector was made fully compatible with Gatewaycloning technology (Thermo Fisher, FIG. 1a ) to facilitate rapidconstruct generation. In order to reduce random cell loss and make thesystem compatible with automated handling/processing steps, we alsogreatly enhanced the adherent properties of our reporter cells to tissueculture plastic, via genomic integration and overexpression of themacrophage scavenger receptor (FIG. 1b ). We also changed our reporterfrom Firefly luciferase to Gaussia princeps luciferase, which has theadvantage of being secreted from cells into the growth media,eliminating the need for a cell lysis step, thus reducing handling stepsand associated variability. Additionally, it produces a significantlyhigher signal than Firefly luciferase (FIG. 1c ), allowing for moresensitive detection.

To test MaMTH-DS sensitivity and its potential suitability for use inRTK drug screening, we selected several RTKs whose dysfunction isassociated with cancer, and prepared stable MaMTH-DS ‘baits’ in ourreporter cell lines. We then performed MaMTH-DS assays in the presenceof transfected Shcl functional adapter protein ‘prey’ and small-moleculeTKIs, including both control molecules and compounds known tospecifically target the function of the corresponding RTKs. First, weexamined the response of the RTK MET to the TKIs Crizotinib andErlotinib using MaMTH-DS. As expected, the interaction was stronglyreduced in a dose-dependent manner when exposed to Crizotinib,consistent with Crizotinib's reported activity against MET, but notErlotinib, which does not target MET (FIG. 2a ). Notably, the responseto Crizotinib was not due to a loss in cell viability (FIG. 3a , LeftPanel), though some reduction in bait expression level was observed(FIG. 3a , Right Panel), suggesting the action of the TKI reduces Metstability (and consequently interaction with Shcl). Next, we tested theresponse of FGFR4 bait to BLU9931, a compound reported to target thisreceptor. Similar to our results with MET, MaMTH-DS reporter activitywas strongly reduced in the presence of BLU9931, but not in the presenceof Erlotinib control (FIG. 2b ). Once again BLU9931had no effect onreporter cell viability (FIG. 3b , Left Panel). An effect of compound onFGFR bait expression was also observed (FIG. 3b , Right Panel), althoughthis was significantly less pronounced than the effect observed withMet. We then proceeded to examine the response of two additional RTKs,AXL and ALK, to the compounds Foretinib and Brigatinib, previously shownto target these receptors, respectively. Once again, both AXL and ALKreporter activity was strongly reduced, in a dose-dependent manner, inthe presence of targeting compound, but not Erlotinib control (FIG. 2cand d ), while cell viability was unaffected (FIG. 3c and d, LeftPanel). Unlike with MET and FGFR4, however, AXL and ALK expression levelwas not altered by compound (FIG. 3c and d, Right Panel), suggestingthat the effect of these TKIs on bait interaction with Shcl is not dueto a global reduction in receptor protein amount/stability.

Example 2

A live-cell, small-molecule screening platform based on the MammalianMembrane Two-Hybrid (MaMTH) was used to screen a collection of 2,960small molecules against an oncogenic Epidermal Growth Factor Receptor(EGFR) mutant resistant to the latest generation of tyrosine kinaseinhibitor therapeutics.

The Mammalian Membrane Two-Hybrid (MaMTH) assay is a technologyspecifically designed for the large-scale identification of theprotein-protein interactions (PPIs) of full-length integral membraneproteins directly in their natural membrane context in live mammaliancells'. MaMTH is able to detect subtle, dynamic changes in PPIs inresponse to mutation state and environmental changes¹⁻³.

Mutants of the Epidermal Growth Factor Receptor (EGFR) are associatedwith cancers such as non-small cell lung cancer (NSCLC) specifically thesingle mutant EGFR L858R, as well as the EGFR/L858R/T790M double andEGFR/L858R/T790M/C797S triple mutants which are associated with acquiredresistance to NSCLC tyrosine kinase inhibitor (TKI) therapeutics⁵⁻⁷. Thedifferential effects of three therapeutic TKIs (Erlotinib, Rociletiniband Osimertinib) on EGFR mutants were assessed by MaMTH using the Shclinteraction partner as prey. In agreement with clinical results, thefirst generation TKI Erlotinib affected the interaction of Shcl with theL858R mutant bait, but not with WT or either drug-resistant mutant,while the third generation TKIs Rociletinib and Osimertinib affectedboth L858R and T790M-carrying mutant baits, but not WT or the C797Striple mutant baits (FIG. 2). The observed differences were not due to areduction in cell viability (FIG. 4a ), and no significant effect onbait expression was evident in response to any of the TKIs (FIG. 4b ).An effect of the TKIs on background MaMTH reporter signal was observedin the absence of prey for EGFR baits (FIG. 4c ) an effect which wasalso observed with FGFR4, AXL and ALK, but not MET (FIG. 5). Furtherinvestigation suggested this was a consequence of a reduction in normalEGFR endosomal trafficking, associated with receptor activation andfunction⁸, in TKI-inhibited mutants (FIG. 6). This could result in lowerbackground reporter activation due to reduced endosomal-mediated EGFRdegradation (and potential non-specific TF release) or EGFR nuclearlocalization⁹. Additionally, this suggests that interaction of EGFR withShcl is affected, at least in part, by TKI-mediated inhibition of mutantEGFR endosomal trafficking, which highlights the ability of MaMTH tosensitively detect loss of functional interactions in response todifferent effects of drug action.

The MaMTH system was modified into a high-throughput, small moleculescreening platform, (MaMTH-DS). MaMTH-DS stably expressed baits,reporter cell lines and MaMTH bait vector construct using the Flp-lNTREx system (Thermo Fisher), a Flp recombinase-based method which allowsfor rapid generation of isogenic stables. The system uses a Gaussiaprinceps luciferase reporter. The system was constructed for use in a384-well format (FIG. 7).

Screening of the EGFR/L858R/T790M/C797S triple mutant was conductedusing MaMTH-DS, which is of great clinical relevance because of itsresistance to the latest generation of clinically approved anti-EGFR TKItherapeutics⁷, against a library of 2960 diverse small-molecules (FIG.8a ). Although drug response with TKIs was observed for sensitivemutants in the absence of prey (FIG. 4c ), screening was performed inthe presence of Shcl, due to the significant enhancement in signal inthe presence of interacting prey (FIG. 9a ), and to allow for theability to detect compounds which might inhibit the interaction of EGFRwith Shcl in a manner not involving an alteration in endosomaltrafficking of the receptor. MaMTH-DS screening was carried out twice(in two independent experiments) to test for reproducibility, and wasperformed in a semi-automated manner, using robotics for cell seeding,sample transfection and small molecule addition. All screen data wassubject to Box-Cox power transformation¹¹ to improve sample datadistribution symmetry and normality prior to further analysis (FIG. 9b). Z-prime values across all ten screened plates exceeded 0.5 in thefirst round of screening (average 0.68 overall), while all ten platesexceeded 0.4 (with seven plates exceeding 0.5) in the second round(average 0.56 overall), supporting excellent assay quality in bothcases¹² (FIG. 8b ). Data normalization was performed on a per platebasis, using both controls-based (Normalized Percent Inhibition, NPI)and sample-based (BScore¹³) approaches, to correct for plate variationand positional effects (FIG. 9c ). NPI and BScore correlated well, andinhibitory hits were scored based on a combined cut-off of greater than70% NPI and a BScore of −3 or less (FIG. 8c ), detecting a total of 49candidates from Round 1 and 45 candidates from Round 2 (FIG. 10).Overlap between both rounds was excellent, with 34 hits shared betweenboth rounds (FIG. 8d ).

To eliminate compounds displaying significant activity against EGFR-WTand/or general toxicity, all 34 shared hits were retested, using MaMTH,in triplicate against both EGFR-L858R-T790M-C797S and EGFR-WT (Table 1).

TABLE 1 Specificity and reproducibility testing of MaMTH-DS hits,identified in both rounds of screening, against EGFR-WT andEGFR-L858R-T790M-C797S baits in the presence of Shcl. Comp. Comp.Average Std. Dev. % CV Average Std. Dev. % CV Source ID Percent PercentPercent Percent Percent Percent OICR TKI OICR0000317A01 129.3 28.5 22.110.5 2.6 24.9 Chembridge 5213777 105.3 30.6 29.1 27.8 11 39.7 OICR TKIOICR0000805A01 80.2 8.1 10.1 18.3 1.2 6.6 OICR TKI OICR0000327B01 57.50.9 1.6 16.3 0.5 2.9 OICR TKI OICR0001145B01 56.4 8.1 14.3 15.8 1.4 8.6Maybridge BR00086SC 24.9 0.5 1.9 6.2 0.4 6.9 Maybridge SPB01851SC 24.55.9 23.8 5.8 1.6 28.1 Maybridge SEW06379SC 23.3 2.1 8.9 17.1 2.6 15.5Chembridge 5304079 21.5 3 13.9 11.7 3.7 31.8 OICR TKI OICR0008718A0119.9 6.3 31.7 4.5 2.3 52.6 Maybridge BTB08928SC 17.3 1.8 10.4 5.3 0.59.9 Chembridge 5106405 15.8 3 18.8 2.7 1.1 41.6 Maybridge S14919SC 13.72.7 19.4 2.9 1.3 44.9 OICR TKI OICR0007886A01 11.5 2.7 23.3 4.6 0.5 9.8OICR TKI OICR0011111A01 7.6 1.9 24.4 0.9 0.2 25.2 Chembridge 5357830 6.22 33.1 0.9 0.2 22 Chembridge 5274945 4.4 0.5 12 0.4 0.1 33.5 MaybridgeKM08160SC 4.1 1.3 32 0.6 0.3 54 OICR TKI OICR0000321A01 2.3 0.4 19.6 0.50.3 71.5 Chembridge 5238658 0.9 0.2 26.7 0.2 0.1 56.1 OICR TKIOICR0011130B01 6.5 2.7 40.9 0.5 0.4 74.7 OICR TKI OICR0000296B01 5.5 2.545.2 0.3 0.2 65.6 OICR TKI OICR0000536B01 7.3 4.1 56.7 0.3 0.1 25.9Maybridge CD03862SC 5.2 2 38.5 2.4 0.8 35.3 Chembridge 5109882 14.8 2.214.6 11.9 1 8.5 Maybridge JA00113SC 3.4 2.3 66.5 0.4 0.1 32.1 OICR TKIOICR0001118A01 4.4 3.4 77.9 0.3 0.2 55.4 OICR TKI OICR0008768A01 6.5 5.788.4 0.3 0 7.5 Chembridge 5792598 84.2 10.2 12.2 114.8 29.8 25.9Chembridge 5333931 45.6 1.8 3.9 42 3.7 8.7 OICR TKI OICR0000531B01 11.50.5 4.6 14.5 3.2 22.3 OICR TKI OICR0011123A01 33.43 8.46 25.3 38.24 1.062.77 Maybridge SEW02515SC 6.7 1.8 26.5 6.4 1.1 17.4 Chembridge 52701408.3 5.2 62.7 8.6 4 46.5 Comp. WT >50% C797S >50% Fold. WT. SourceInhibition Inhibition p-Value C797S OICR TKI NO YES 0.017991867 12.3Meet Chembridge NO YES 0.036213511 3.8 criteria OICR TKI NO YES0.004903998 4.4 for OICR TKI NO YES 7.30E−06 3.5 establishing OICR TKINO YES 0.011228412 3.6 reproducibility and mutant specificity. MaybridgeYES YES 9.30E−07 4 Do not Maybridge YES YES 0.024156361 4.3 meetMaybridge YES YES 0.035165788 1.4 criteria Chembridge YES YES0.025512837 1.8 for OICR TKI YES YES 0.038481202 4.5 establishingMaybridge YES YES 0.004433972 3.2 reproducibility Chembridge YES YES0.009345886 5.8 and mutant Maybridge YES YES 0.008764106 4.8specificity. OICR TKI YES YES 0.043699193 2.5 OICR TKI YES YES0.023346445 8.1 Chembridge YES YES 0.046079898 6.6 Chembridge YES YES0.004005023 12 Maybridge YES YES 0.037344862 6.6 OICR TKI YES YES0.006113406 5 Chembridge YES YES 0.022392801 4.4 OICR TKI YES YES0.057112926 12.4 OICR TKI YES YES 0.068205877 16.3 OICR TKI YES YES0.098676818 26.6 Maybridge YES YES 0.120873261 2.2 Chembridge YES YES0.1303195  1.2 Maybridge YES YES 0.146340884 8.9 OICR TKI YES YES0.175813511 13.1 OICR TKI YES YES 0.203901998 20 Chembridge NO NO0.209695268 0.7 Chembridge YES YES 0.221411804 1.1 OICR TKI YES YES0.239198175 0.8 OICR TKI YES YES 0.429149913 0.9 Maybridge YES YES0.830386676 1 Chembridge YES YES 0.931181228 1 *C797S refers to thetriple mutant

From these results, compounds were selected for further considerationonly if they inhibited EGFR/L858R/T790M/C797S (but not EGFR-WT) greaterthan 50% and if the difference in their inhibition of mutant vs WT wasboth statistically significant and at least 2-fold (FIG. 8e and Table1). The 5 compounds satisfying these criteria were then subjected todose-response testing (FIG. 11a and b ), of which 3 were found todisplay robust, dose-dependent inhibition of EGFR-L858R-T790M-C797S,meeting the above criteria for at least two different doses (FIG. 8e andFIG. 11a ).

These 3 final candidates included the Chembridge Diverset compound5213777 (3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one), andthe TKIs AZD7762 (OICR0001145B01) and Midostaurin (OICR0000317A01).There is little published information available for3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and it is anexample of a coumarin derivative.7-Diethylamino-3(2′-benzoxazolyl)-coumarin has been reported as amicrotubule inhibitor with antimitotic activity in multidrug resistantcancer cell lines²⁵. Its specificity in the assay does not appear to bea consequence of any general activity against the viability of thereporter cell constructs used in the screen, however, as it has nosubstantial effect on the viability of either EGFR WT or EGFRL858R/T790M/C797S expressing cells (FIG. 12). In contrast to3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, both TKIshave been well-studied. AZD7762 is a CHK1/2 kinase inhibitor, and hasnot been previously reported as active against EGFR NSCLC mutants. Ithas been the subject of a variety of studies, including a Phase IClinical trial for the treatment of cancerous solid tumors^(14,15).Midostaurin (OICR0000317A01), is a multi-kinase inhibitor that has beenrecently approved by the FDA for use in the treatment of FLT3-mutantacute myeloid leukemia (AML)¹⁶. It was previously investigated for useagainst EGFR-L858R-T790M double mutant¹⁷, however its activity has notbeen shown against EGFR-L858R-T790M-C797S triple mutant.

Further examination of these 3 final candidates was carried out in Ba/F3cells expressing either EFGR-WT, EGFR-L858R-T790M-C797S orEGFR-ex19del-T790M-C797S (another common oncogenic variant of EGFR)(FIG. 13, FIGS. 14 and 15). Notably, all compounds had an effect on thephosphorylation and/or expression of EGFR and downstream signallingmolecules in both mutant lines, but not in WT, with the strongestresponse observed for Midostaurin (FIG. 13a and FIGS. 14a and 15a ).Midostaurin also caused a robust, dose-dependent reduction in theviability of Ba/F3 cells expressing mutant EGFR, much stronger thanobserved for WT (FIG. 13b ), while3-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one caused only amild/modest reduction in cell viability of all three lines (with thegreatest effect on EGFR-WT) (FIG. 14b ), and AZD7762 had no strongeffect on the viability of any of the cell lines tested (FIG. 15b ).Strikingly, the potency of all three compounds against cell viabilitywas significantly enhanced in the presence of the therapeutic anti-EGFRantibodies Cetuximab and Panitumumab in both EGFR-L858R-T790M-C797S(FIG. 13c , FIG. 14c and FIG. 15c ) and EGFR-ex19del-T790M-C7975 (FIG.13d , FIG. 14d and FIG. 15d ) triple mutants, similar to the behavior ofBrigatinib (FIG. 13c and d ), a TKI recently shown to be effectiveagainst EGFR-C797S triple mutants alone and in combination withtherapeutic antibody¹⁸. Interestingly, however, all three of ourcompounds displayed a notably enhanced potency against L858R triplemutant in the presence of Cetuximab that was not observed forBrigatinib, suggesting the compounds may have broader efficacy (compareFIG. 13c , FIG. 14c and FIG. 15c ). Importantly, no such enhancement wasevident using AZD9291 (Osimertinib), which does not target EGFR C797Smutants, as a control (FIG. 16).

To obtain more information about the specific mechanism of action of thecompounds against EGFR we next performed traditional in vitro kinaseassays using recombinant kinase domain from the EGFR WT, EGFRex19del/T790M/C797S and EGFR L858R/T790M/C797S proteins. In theseassays, we observed that Midostaurin strongly inhibited the kinaseactivity of the EGFR triple mutants, but had no significant effect onEGFR WT activity, even at a concentration of 10 micromolar (FIG. 17a ).This was in sharp contrast to Brigatinib, which although it was also apotent inhibitor of triple mutant, had a significant effect on EGFR WTactivity, with an IC50 of ˜141 nanomolar (FIG. 17b ). AZD7762 alsodisplayed expected kinase inhibition of EGFR triple mutants, with IC50sof ˜89.3 and 282 nanomolar for the ex19del and L858R triple mutants,respectively, however, strikingly, its activity against the EGFR L858Rmutant was comparable to that of WT (IC50 of ˜258 nanomolar) (FIG. 17c). This is an intriguing result, suggesting that the specificity of thecompound for EGFR L858R triple mutant over WT detected in our assay is areflection of the use of the full-length protein and/or other conditionspresent only in a live-cell format, highlighting a potential strength ofour system over traditional in vitro kinase assays, which would not haveidentified this specificity. Finally,3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one displayed nokinase inhibitory activity against EGFR WT or either of the EGFR triplemutants (FIG. 17d ), consistent with the fact that it has no previouslyreported activity against kinases, and lacks a classic kinase inhibitorpharmacophore. While the mechanism of action of this compound isunclear, its detection in our MaMTH-DS assay using EGFRL858R/T790M/C797S as bait, its effect on the phosphorylation of EGFR anddownstream signalling molecules in Ba/F3 cells expressing both EGFRex19del/T790M/C797S and L858R/T790M/C797S triple mutants (but not WT),and its enhanced potency against triple mutant Ba/F3 cells in thepresence of anti-EGFR therapeutic antibodies, clearly supports an EGFRspecific involvement. Exactly how this compound targets EGFR mutant,including possible roles as an allosteric inhibitor or a directdisruptor or EGFR interactions, will be the focus of future studies.

MaMTH as demonstrated herein can sensitively detect inhibitory compoundsthat change the phosphorylation status of full-length EGFR proteins inthe context of living cells and in the low nanomolar range, with thebenefit that identified small molecule candidates have already passedcell permeability and toxicity tests.

Methods

MaMTH assays. Cells stably expressing bait of interest (EGFR, MET,FGFR4, ALK, AXL) were seeded into 96-well TC-treated plates and grown at37° C./5% CO₂ overnight in DMEM/10% FBS/1% PS to ˜50-60% confluency.Cells were transfected with 50 ng/well of Nub-Shc1 ‘prey’ protein bycalcium phosphate precipitation. Five hours after transfection, mediawas aspirated out and cells were treated with 100 uL of fresh mediacontaining specific compound and 0.5 ug/ml of tetracyline to induce baitexpression. After 24 hours, luciferase activity was measured bychemoluminescence.

Western analysis of bait and downstream signalling molecule expressionand phosphorylation. Cells grown under the specified conditions werewashed with ice cold PBS before addition of the cell lysis buffer (CellLysis Buffer 10×, Cell Signalling Technology, #9803) supplemented withprotease inhibitors. Lysates were transferred to 1.5 mL microtubes, andcentrifuged for 15,000 rpm for 10 min. The supernatants were mixed withLaemmli sample buffer, and boiled at 95° C. for 5 min. Proteinquantification was performed using the BCA Protein Assay Reagent(Pierce) according to the manufacturer's protocol prior to addition ofsample buffer. Western blot analyses were performed after separation bySDS-PAGE, and transferred to nitrocellulose membranes. The membraneswere then blocked with 2% BSA in Tris-buffered saline/Tween 20 (TBS-T).Antibodies used for Western blot analysis were: phospho-EGFR antibody(Tyr1173; Santa Cruz, sc101668, 1:10,000), total EGFR (Cell signallingTechnology, #4267, 1:10,000), phospho-AKT (Ser473; Cell SignallingTechnology, #4060, 1:10,000), total AKT (Cell signalling Technology,#4691, 1:10,000), phospho-ERK (Thr202/Tyr204; Cell SignallingTechnology, #9101, 1:10,000), total ERK1/2 (Cell Signalling Technology,#9102, 1:10,000), phospho-S6 (Ser240/244, Cell Signalling Technology,#5364, 1:10,000), total S6 (Cell Signalling Technology, #5364,1:10,000), anti-GAPDH (Santa Cruz, 1:10,000), anti-tubulin (Santa Cruz,1:10,000) or anti-V5 (Cell Signalling Technology, 1:10,000).

Cell viability assays. MaMTH stable bait cells or Ba/F3 cells wereseeded into 96 well plates at 10,000 cells per well. For MaMTH stablecells, the cells were treated the next day with each inhibitor in adose-dependent manner in addition to 0.5 ug/mL tetracyline to inducebait expression. For BaF3 cells, the cells were treated with eachcompound the same day as seeding. After 72 hours of drug treatment, cellviability was measured using the CellTiter-Glo assay (Promega).

EGFR localization and trafficking analyses. The experiment was performedin 384 well CellCarrier imaging plates. Each condition (EGF stimulationtime, treatment and mutation) was repeated in at least 6 wells. 11images (276×234 μm) were collected from each well by automated confocalmicroscope CV7000 (Yokogawa) with a 60× water immersion objective(NA=1.2), with a total of 1063±251 (mean±SD) imaged cells per well.Images were analyzed by MotionTracking software(http://motiontracking.mpi-cbg.de) and 147±80 (mean±SD) EEA1-positiveendosomes per cell were found. All statistics were calculated per image,then averaged between images in the well, and, finally, averaged betweenwells of equal conditions. The SEM was calculated from the lastaveraging step.

Generation of adherent HEK293 cells. Flp-ln 293 TREx cells (ThermoFisher) were grown at 37° C./5% CO₂ in DMEM/10% FBS/1% PS media in6-well TC-treated plates to ˜50-60% confluency. Cells were thentransfected with pcDNA3.1 plasmid, expressing the gene for humanMacrophage Scavenger Receptor 1 (MSR1) transcript variant A alongsideG418 resistance cassette, using PolyJet transfection reagent (SignaGen),as per manufacturer instructions. Cells were grown overnight and thensplit into 10 cm plate containing 10 mL of DMEM/10% FBS/1% PS/800 ug/mLG418 and grown at 37° C./5% CO₂ until distinct foci appeared. Individualfoci were expanded, and screened for enhanced adherence using methyleneblue staining and stringent washing in a 96-well plate format aspreviously described¹⁰. The most highly adherent cell line displayingrobust growth in media and appropriate Flp-ln 293 TREx resistance toZeocin and Blasticidin was selected for use in the generation of MaMTHreporter cells.

Generation of stable MaMTH reporter cells. Reporter vector was generatedin a pcDNA3.1(−) backbone using ORFs expressing Gaussia princepsluciferase (New England Biolabs) under the control of a 5×GAL4 UAS andpuromycin resistance marker under the control of a constitutive PGKpromoter, via Gibson assembly¹⁹. Adherent FLP-compatible HEK293 cells(prepared above) were grown at 37° C./5% CO₂ in DMEM/10% FBS/1% PS mediain 6-well TC-treated plates to ˜50-60% confluency. Cells weretransfected with 1000 ng reporter vector using X-tremeGENE 9 DNAtransfection reagent (Roche) as per manufacturer instructions. After 5hours, media containing transfection reagent was removed and replacedwith fresh DMEM/10% FBS/1% PS. Cells were grown for 48 hours and thensplit 1 in 2 into new 6-well plates using DMEM/10% FBS/1% PS+0.5 ug/mLpuromycin and grown until individual foci appeared. Individual foci wereexpanded and monoclonal populations isolated by sorting of individualcells into 96-well plates using a FACS Aria II Flow Cytometer (BDBiosciences), followed by further expansion. Expanded cell populationswere screened individually and a cell line displaying strongMaMTH-responsive reporter activity and minimal background was selectedfor further use in MaMTH-DS.

Generation of Flp-ln TREx compatible MaMTH bait vectors. Gateway-cloningcassette followed by Cub-GAL4/RelA TF sequence was PCR-amplified off ofour previously reported MaMTH bait vector¹ using KAPA 2× HiFi DNAPolymerase (Kapa Biosystems). Amplified fragment was combined withEcoRV-digested Flp-compatible pcDNA5/FRT/TO vector (Thermo Fisher) viaGibson Assembly¹⁹. Generated constructs were fully sequenced verified,and construct containing all of the elements necessary for GatewayCloning, tetracycline-induction, MaMTH bait C-tagging and use ingeneration of isogenic stables via the Flp-ln TREx systemtetracyline-incudible, was isolated. This final bait vector constructwas designated A1160.

Generation of Flp-ln TREx compatible MaMTH bait constructs. All bait andprey constructs were generated using the Gateway cloning technology(Thermo Fisher) and destination vectors A1160 (MaMTH bait) or A1245(MaMTH prey). Shcl ORF in entry clone format was obtained from the HumanORFeome Collection V8.1²⁰. EGFR-WT and single L858R and doubleL858R/T790M mutant entry clones were generated as described previously¹.EGFR triple mutant containing the C797S mutation was generated viasite-directed mutagenesis of EGFR double mutant using primers5′-atgcccttcggcagcctcctggact-3′ and 5′-agtccaggaggctgccgaagggcat-3′ (SEQID NO: 1 and 2). MET entry clone was obtained from OpenFreezer (V9936).All final bait and prey constructs were fully sequence verified.

Generation of stable MaMTH bait cell lines. Isogenic MaMTH reporter celllines stably expressing baits of interest were generated using theFlp-ln TREx system (Thermo Fisher). Briefly, MaMTH reporter cells weregrown at 37° C./5% CO₂ in DMEM/10% FBS/1% PS media in 6-well TC-treatedplates to ˜50-60% confluency. Cells were transfected with 900 ng pOG44and 100 ng of bait construct in A1160 using X-tremeGENE 9 DNAtransfection reagent (Roche) as per manufacturer instructions. After 5hours, media containing transfection reagent was removed and replacedwith fresh DMEM/10% FBS/1% PS. Cells were grown for 48 hours and thensplit 1 in 2 into new 6-well plates using DMEM/10% FBS/1% PS+100 ug/mLHygromycin and grown until individual foci appeared. Foci were expandedand proper, tetracycline-induced bait expression was verified by Westernblotting.

MaMTH-DS high-thoughput screening workflow. MaMTH reporter cells stablyexpressing EGFR/L858R/T790M/C797S bait were seeded into 384-well plates(5000 cells/well) in DMEM/10% FBS/1% PS media using a MultiDrop Combi(Thermo) fitted with a standard cassette. Plates were covered withMicroClime Environmental Lids (Labcyte; hydrated with ˜10 mL ddH2O) andgrown at 37° C./5% CO₂ overnight. The next day cells were transfectedwith 25 ng of MaMTH Shcl prey DNA using X- X-tremeGENE 9 DNAtransfection reagent (Roche) as per manufacturer instructions.Transfection mix (5 uL total volume/well) was added to 384-well platescontaining cells using a Bravo Automated Liquid Handling Platform(Agilent) fitted with a 96ST pipette head. Plates were once againcovered with MicroClime Lids and grown at 37° C./5% CO₂ for 5 hours.Media was then removed from plates using a BioTek 405 Select microplatewasher, and a fresh 50 uL of DMEM/10% FBS/1% PS media containing 0.5ug/mL Tetracycline was added to each well using a MultiDrop Combi. 50 nLof DMSO, AZD9291 (70 uM) or library compound (10 mM forChembridge/Maybridge compounds, 1 mM for TKIs were then added toindividual wells using an ECHO 550 (Labcyte) (the final concentration offor the Chembridge/Maybridge compounds was 10 uM and 1 uM for the TKIs).Plates were covered with MicroClime lids and grown for an additional17-18 hours at 37° C./5% CO₂. Cells were then subjected to luciferaseassay using 20 uL of 20 uM coelenterazine per well. Luminescence wasmeasured in an injector-equipped SynergyNeo microplate reader, usinglinear shaking for 2 seconds after substrate addition. All reads wereperformed from the top using a Gain of 100 and a 1 second integrationtime.

Data analysis of MaMTH-DS screening results. All data was analysis wasperformed in an automated fashion using in house-software developed inthe R programming language²¹. Raw data from screens were subjected toBox-Cox transformation as previously described¹¹ in order to improvedata distribution symmetry and normality. Z-prime¹² values werecalculated on a per plate basis using EGFRL858R-T790M andEGFR-L858R-T790M-C797S in the presence of AZD9291 as positive andnegative controls respectively (with the exception of Shcl Round 2 Plate10, where, due to a technical issue, EGFR-L858R-T790M-C797S in thepresence of DMSO was used as a negative control instead). Prior to Z′calculations, the single most extreme value from each control datasetwas excluded if it was classified as a outlier based on a cut-off of 1.5times the IQR. Data normalization was performed using bothcontrols-based Normalized Percent Inhibition (NPI) and sample-based(controls independent) BScore. NPI was calculated as (Negative ControlSignal Sample Signal)/(Negative Control Signal Positive ControlSignal)*100. B-Score was calculated using the cellHTS2 package²². NPIwas plotted against BScore and hits were scored using a combined cut-offof 70% NPI and a BScore of −3 or less.

In vitro Kinase assays. Kinase assays were performed using recombinantproteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R,EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).Compounds (Midostaurin, AZD7762, Chembridge 5213777 and Brigatinib) weretested in a 10-dose IC50 duplicate mode with 3-fold serial dilutionstarting at 10 μM. Reactions were carried out at 10 μM ATP.

Example 3

Further examination of the effects of midostaurin on mutant EGFRactivity was carried out. In vitro kinase assays demonstrated that thatmidostauin inhibits the kinase activity of EGFR double and triplemutants (FIG. 18a ). Examination of the effects of midostaurin on mutantEGFR activation and downstream signaling was carried out in PC9 cellsexpressing EGFR-ex19del-T790M-C797S (FIGS. 18b and 19). Midostaurin hadan inhibitory effect on the phosphorylation and/or expression of EGFRand downstream signaling molecules (FIG. 18b ). Midostaurin caused adose-dependent activation of caspase 3 and 7 activity that was notobserved either in CFBE cells or in response to osimertinib (FIG. 19a ).Midostaurin also caused a dose-dependent reduction in viability of PC9organoids expressing EGFR-ex19del-T790M-C797S that was not observed forosimertinib (FIG. 19b ).

Further examination of the effects of3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one was carriedout. The ability of3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one to inhibitcomplex formation was tested using MaMTH (FIG. 20a and b ).3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one blockedcomplex formation between Shc1 and EGFR L858R/T790M/C797S but notwild-type EGFR or the other tested RTKs (FIG. 20a ).3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, but notosimertinib, blocked complex formation between EGFR L858R/T790M/C797Sand Shc1, Crkll, and Hsp90 in a dose-dependent manner (FIG. 20b ). Invitro kinase assays demonstrated that that3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one does notinhibit the kinase activity of EGFR triple mutants (FIG. 20c ).Examination of the effects of3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one on mutant EGFRactivation and downstream signaling was carried out in PC9 cellsexpressing EGFR-ex19del-T790M-C797S (FIGS. 20d and 21).3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one had aninhibitory effect on the phosphorylation and/or expression of EGFR anddownstream signaling molecules (FIG. 20d ).3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one caused adose-dependent decrease in viability in PC9 cells expressingEGFR-ex19del, EGFR-ex19del-T790M, or EGFR-ex19del-T790M-C797S mutantsthat was not observed either in CFBE cells expressing wild-type EGFR(FIG. 21a ). 3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-onecaused a dose-dependent activation of caspase 3 and 7 activity in PC9cells expressing EGFR-ex19del-T790M-C797S mutants that was not observedeither in CFBE cells or in response to osimertinib (FIG. 21b ).3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one also caused adose-dependent reduction in viability of PC9 organoids expressingEGFR-ex19del-T790M-C797S that was not observed for osimertinib (FIG. 19b).

Briefly, PC9 EGFR ex19del/T790M/C797S organoids were developed by thePrincess Margaret Living Biobank Organoid Center. Briefly, cells wereadapted to grow in a Matrigel culture and were seeded into 384 wellplates for the cell viability assay. Organoids were allowed to grow for72 hours with the compound, and cell viability was measured usingCellTitre glow cell viability reagent.

Example 4

The success of midostaurin in the above examples prompted a search forother molecules with similar activity. Gilteritinib is another FLT3inhibitor that has been proposed for use in Acute Myeloid Leukemiapatients harboring a FLT3 mutation. Gilteritinib was therefore testedfor the ability to inhibit EGFR mutants.

The ability of gilteritinib to inhibit EGFR complex formation was testedusing MaMTH (FIG. 22a ). Gilteritinib inhibited complex formationbetween Shc1 and EGFR mutants L858R/T790M and L858R/T790M/C797S, but notwild-type EGFR, in a dose-dependent maner. In vitro kinase assaysdemonstrated that that gilteritinib preferentially inhibits the kinaseactivity of EGFR ex19del-T790M, ex19del-T790M-C797S, andL858R/T790M/C797S, with the most robust inhibition demonstrated forex19del-T790M-C797S (FIG. 22b ). Examination of the effects ofgilteritinib on mutant EGFR activation and downstream signaling wascarried out in PC9 cells expressing EGFR-ex19del-T790M-C797S (FIGS. 22cand 23). Gilteritinb had an inhibitory effect on the phosphorylationand/or expression of EGFR and downstream signaling molecules (FIG. 22c). Gilteritinib caused a dose-dependent reduction in viability of PC9organoids expressing EGFR-ex19del-T790M-C797S that was not observed forosimertinib (FIG. 23). The potency gilteritinib against cell viabilitywas significantly enhanced in the presence of the therapeutic anti-EGFRtherapeutic antibody Panitumumab EGFR-ex19del-T790M-C797S triplemutants, similar to the behavior of midostaurin (FIG. 23b ).

Briefly, PC9 EGFR ex19del/T790M/C797S cells were seeded into 96 wellplates and treated with 10 ug/ml Panitumumab in combination withdifferent doses of Gilteritinib or Midostaurin. Cells were allowed togrow for 72 hours before measuring cell viability using CellTitre bluereagent.

Example 5 Analog and Combination Treatment

FIG. 24 shows the dose response analysis from MaMTH-DS screens of EGFRL858R/T790M/C797S in the presence of Shcl, and the PC9 cell viabilityresults for 5213777 and a chlorine substituted analog. Also shown is abenzoxazolyl-chromen-2-one compound which did not show does response ortoxicity for comparison.

As shown in FIG. 25, Gilteritinib (a) or Midostaurin (b) in combinationwith 50 nM of compound 5213777 has an increased effect at reducing thecell viability of PC9 EGFR ex19del/T790M/C797S cells after 72 hourscompared to Gilteritinib or Midostaurin alone. No combination effect wasobserved with the benzoxazolyl-chromen-2-one compound which did not showtoxicity when added on its own to cells overexpressing EGFRex19del/T790M/C797S.

Example 6

Biopsies from patients with lung cancer are tested for the presence ofthe EGFR/L858R/T790M double or EGFR/L858R/T790M/C797S triple mutations.Patients that test positive, are then will be administered with atherapeutically effective amount of gilteritinib in combination with atherapeutically effective amount of one or more of the compounds offormula I, II, or III of the disclosure and/or an EGFR therapeuticantibody.

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

The scope of the claims should not be limited by the preferredembodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   -   1. Petschnigg, J. et al. The mammalian-membrane two-hybrid assay        (MaMTH) for probing membrane-protein interactions in human        cells. Nat. Methods 11, 585-92 (2014).    -   2. Yao, Z. et al. A Global Analysis of the Receptor Tyrosine        Kinase-Protein Phosphatase Interactome. Mol. Cell 65, 347-360        (2017).    -   3. Petschnigg, J. et al. Systematic Identification of Oncogenic        EGFR Interaction Partners. J. Mol. Biol. 429, 280-294 (2017).    -   4. Goździk-Spychalska, J. et al. C-MET inhibitors in the        treatment of lung cancer. Curr. Treat. Options Oncol. 15, 670-82        (2014).    -   5. da Cunha Santos, G., Shepherd, F. A. & Tsao, M. S. EGFR        Mutations and Lung Cancer. Annu. Rev. Pathol. Mech. Dis. 6,        49-69 (2011).    -   6. Yu, H. A. et al. Analysis of Tumor Specimens at the Time of        Acquired Resistance to EGFR-TKI Therapy in 155 Patients with        EGFR-Mutant Lung Cancers. Clin. Cancer Res. 19, 2240-2247        (2013).    -   7. Thress, K. S. et al. Acquired EGFR C797S mutation mediates        resistance to AZD9291 in non-small cell lung cancer harboring        EGFR T790M. Nat. Med. 21, 560-2 (2015).    -   8. Jones, S. & Rappoport, J. Z. Interdependent epidermal growth        factor receptor signalling and trafficking. Int. J. Biochem.        Cell Biol. 51, 23-28 (2014).    -   9. Lin, S. Y. et al. Nuclear localization of EGF receptor and        its potential new role as a transcription factor. Nat. Cell        Biol. 3, 802-8 (2001).    -   10. Robbins, A. K. & Horlick, R. A. Macrophage scavenger        receptor confers an adherent phenotype to cells in culture.        Biotechniques 25, 240-4 (1998).    -   11. Box, G. E. P. and Cox, D. R. An analysis of        transformations. R. Stat. Soc 26, 211-252 (1964).    -   12. Zhang, J.-H., Chung & Oldenburg. A Simple Statistical        Parameter for Use in Evaluation and Validation of High        Throughput Screening Assays. J. Biomol. Screen. 4, 67-73 (1999).    -   13. Brideau, C., Gunter, B., Pikounis, B. & Liaw, A. Improved        Statistical Methods for Hit Selection in High-Throughput        Screening. J. Biomol. Screen. 8, 634-647 (2003).    -   14. Zabludoff, S. D. et al. AZD7762, a novel checkpoint kinase        inhibitor, drives checkpoint abrogation and potentiates        DNA-targeted therapies. Mol. Cancer Ther. 7, 2955-2966 (2008).    -   15. Sausville, E. et al. Phase I dose-escalation study of        AZD7762, a checkpoint kinase inhibitor, in combination with        gemcitabine in US patients with advanced solid tumors. Cancer        Chemother. Pharmacol. 73, 539-549 (2014).    -   16. Levis, M. Midostaurin approved for FLT3-mutated AML. Blood        129, 3403-3406 (2017).    -   17. Lee, H.-J. et al. Noncovalent wild-type-sparing inhibitors        of EGFR T790M. Cancer Discov. 3, 168-81 (2013).    -   18. Uchibori, K. et al. Brigatinib combined with anti-EGFR        antibody overcomes osimertinib resistance in EGFR-mutated        non-small-cell lung cancer. Nat. Commun. 8, 14768 (2017).    -   19. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up        to several hundred kilobases. Nat. Methods 6, 343-345 (2009).    -   20. Yang, X. et al. A public genome-scale lentiviral expression        library of human ORFs. Nat. Methods 8, 659-61 (2011).    -   21. R-Core-Team. R: A language and environment for statistical        computing. (2017). at <www.R-project.org>    -   22. Boutros, M., Ligia, P., Bras, L. & Huber, W. Analysis of        cell-based RNAi screens. Genome Biol. 7, R66 (2006).    -   23. Lee, H J et al. EGFR T790M-Selective Indolocarbazole        Compounds. Cancer Discov 3, 168-181(2013)    -   24. Costa D B and Kobayashi Transl Lung Cancer Res 4: 809-815        (2015)    -   25. Kim, S-N. et al. 7-Diethylamino-3(2′benzoxazolyl)-coumarin        is a novel microtubule inhibitor with antimitotic activity in        multidrug resistant cells. Biochem. Pharmacol. 77, 1773-1779.

1.-29. (canceled)
 30. A composition or combination comprising at leasttwo compounds selected from:3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one and/orstructurally related analog thereof, the structurally related analogthereof selected from benzoxazolyl and/or chromenone containingcompounds, substituted versions thereof or a salt or solvate of any ofthe foregoing; midostaurin or a salt or solvate thereof; gilteritinib ora salt or solvate thereof;3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide;and an anti-EGFR therapeutic antibody.
 31. The composition orcombination of claim 30, wherein the composition or combinationcomprises: a) i)3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, astructurally related analog thereof, the structurally related analogthereof selected from benzoxazolyl and/or chromenone containingcompounds, substituted versions thereof or a salt or solvate of any ofthe foregoing, ii) midostaurin or a salt or solvate thereof, iii)gilteritinib or a salt or solvate thereof, or iv)3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide;and b) an anti-EGFR therapeutic antibody.
 32. The composition orcombination of claim 30, wherein the composition is a pharmaceuticalcomposition.
 33. The composition or combination of claim 32, wherein thecomposition is in a dosage form selected from a solid dosage form and aliquid dosage form.
 34. The composition or combination of claim 32,wherein the composition is an injectable dosage form.
 35. Thecomposition or combination of claim 32, wherein the composition isformulated for administration by parenteral, intravenous, subcutaneous,intracardial, intramuscular, or oral administration. 36.-43. (canceled)44. The composition or combination of claim 30, wherein the structurallyrelated analog thereof is a halogen substituted benzoxazolyl analog orchlorine substituted analog thereof.
 45. The composition or combinationof claim 37, wherein the chlorine substituted analog is3-(5-chloro-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one. 46.The composition or combination of claim 30, wherein the chromenonecontaining compound is selected from the following chromen-2-onederivatives: 7-Methoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;7-Ethoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;3-(2-[4-(Chromen-2-one-3-yl)tiazol-2-yl]thia-zol-4-yl)-chromen-2-one;7-Methoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;7-Hydroxy-3-(2-phenyl-thiazol-4-yl)-chro-men-2-one;6-Methoxy-3[4(4-methoxy-phenyl)thia-zol-2-yl]-chromen-2-one;7-Hydroxy-3-[4-(4-methoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;7-Diethylamino-3-[2-(4-dimethylamino-phenyl)-thiazol-4-yl]-chromen-2-one;3-[4-(4-Chloro-phenyl)-thiazol-2-yl]-6-hexyl-7-hydroxy-chromen-2-one;6-Hexyl-7-hydroxy-3-[4-(4-phenoxy-phe-nyl)-thiazol-2-yl]-chromen-2-one;7-Hydroxy-3-[4-(4-phenoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;6-Hexyl-7-hydroxy-3-[4-(4-methoxy-phe-nyl)-thiazol-2-yl]-chromen-2-one;7-Diethylamino-3-(4-methyl-thiazol-2-yl)-chro-men-2-one;3-[4-(4-Bromo-phenyl)-thiazol-2-yl]-6-hex-yl-7-hydroxy-chromen-2-one;6-Hexyl-7-hydroxy-3-(2-phenyl-thia-zol-4-yl)-chromen-2-one,6-Hexyl-7-hydroxy-3-(4-phenyl-thiazol-2-yl)-chromen-2-one;7-Hydroxy-3-(4-phenyl-thiazol-2-yl)-chromen-2-one;6-Hexyl-7-hydroxy-3-(4-methyl-thiazol-2-yl)-chromen-2-one;7-Hydroxy-3-(5-methyl-4-phenyl-thiazo-2-yl)-chromen-2-one;3-[4-(4-Chloro-4-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;7-Hydroxy-3-(5-methyl-4-p-tolyl-thiazol-2-yl)-chromen-2-one;6-Bromo-3-[4-(4-ethoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;3-[4-(4-Ethyl-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[4-(4-Chloro-phenyl)-thiazol-2-yl]-6-meth-oxy-chromen-2-one;3-[2-(3,4-Dimethoxy-phenyl)-thiazol-4-yl]-chro-men-2-one;3-[4-(4-Bromo-phenyl)-5-ethyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-(4,5-Dihydro-naphtho[1,2-d]thiazol-2-yl)-7-hy-droxy-chromen-2-one;7-Diethylamino-3-(4-phenyl-thiazol-2-yl)-chro-men-2-one;3-[4-(3,4-Dichloro-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[4-(4-Chloro-phenyl)-5-ethyl-thia-zol-2-yl]-7-hydroxy-chromen-2-one,3-(5-Ethyl-4-p-tolyl-thiazol-2-yl)-7-hy-droxy-chromen-2-one;7-Diethylamine-3-(2-phenyl-thiazol-4-yl)-chro-men-2-one;3-[4-(2,5-Dimethyl-phenyl)-5-ethyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[2-(4-Hydroxy-phenyl)-thiazol-4-yl]-chro-men-2-one;3-(5-Ethyl-4-phenyl-thiazol-2-yl)-7-hy-droxy-chromen-2-one;3-[2-(2,4-Dimethyl-phenyl)-thiazol-4-yl]-7-hy-droxy-chromen-2-one;3-[4-(3-Bromo-phenyl)-thazol-2-yl]-8-meth-oxy-chromen-2-one;7-Hydroxy-3-[4-(3-methoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;3-Benzothiazol-2-yl-7-hydroxy-chro-men-2-one;3-(5-Chloro-1H--benzoimidazol-2-yl)-8-meth-oxy-chromen-2-one;3-[5-(3-Fluoro-phenyl)-[1,3,4]oxa-diazol-2-yl]-8-methoxy-chromen-2-one;3-Benzo[d]imidazo[2,1-b]thiazol-2-yl-chro-men-2-one;3-(7-Methoxy-benzo[d]imidazo[2,1-b]thia-zol-2-yl)-chromen-2-one;6-Chloro-3-(7-fluoro-benzo[d]imi-dazo[2,1-b]thiazol-2-yl)-chromen-2-one;3-Benzo[d]imidazo[2,1-b]thiazol-2-yl-6-hex-yl-7-hydroxy-chromen-2-one;6,8-Dichloro-3-(5-p-tolylamino-[1,3,4]thia-diazol-2-yl)-chromen-2-one;3-[5-(3-Chloro-phenyl)-[1,3,4]oxa-diazol-2-yl]-6-hexyl-7-hydroxy-chro-men-2-one;6-Hexyl-7-hydroxy-3-[5-(3-methoxy-phe-nyl)-[1,3,4]oxadiazol-2-yl]-chro-men-2-one;6-Hexyl-7-hydroxy-3-(7-methyl-imidazo[1,2-a]pyridin-2-yl)-chromen-2-one;6-Methoxy-3-[2-(4-methoxy-phenyl-amino)-thiazol-5-yl]-chromen-2-one; and6-hexyl-7-hydroxy-3-[5-(3,4,5-trimeth-oxy-phenyl)-[1,3,4]oxadiazol-2-yl]-chro-men-2-one.47. The composition or combination of claim 30, wherein the anti-EGFRtherapeutic antibody is cetuximab or an antigen-binding fragment thereofor panitumumab or an antigen-binding fragment thereof.
 48. Thecomposition or combination of claim 31, wherein the composition is apharmaceutical composition.
 49. The composition or combination of claim41, wherein the composition is in a dosage form selected from a soliddosage form and a liquid dosage form.
 50. The composition or combinationof claim 41, wherein the composition is an injectable dosage form. 51.The composition or combination of claim 41, wherein the composition isformulated for administration by parenteral, intravenous, subcutaneous,intracardial, intramuscular, or oral administration.
 52. The compositionor combination of claim 41, wherein the structurally related analogthereof is a halogen substituted benzoxazolyl analog or chlorinesubstituted analog thereof.
 53. The composition or combination of claim45, wherein the chlorine substituted analog is3-(5-chloro-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one. 54.The composition or combination of claim 31, wherein the chromenonecontaining compound is selected from the following chromen-2-onederivatives: 7-Methoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;7-Ethoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;3-(2-[4-(Chromen-2-one-3-yl)tiazol-2-yl]thia-zol-4-yl)-chromen-2-one;7-Methoxy-3-(2-methyl-thiazol-4-yl)-chro-men-2-one;7-Hydroxy-3-(2-phenyl-thiazol-4-yl)-chro-men-2-one;6-Methoxy-3-[4-(4-methoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;7-Hydroxy-3-[4-(4-methoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;7-Diethylamino-3-[2-(4-dimethylamino-phenyl)-thiazol-4-yl]-chromen-2-one;3-[4-(4-Chloro-phenyl)-thiazol-2-yl]-6-hexyl-7-hydroxy-chromen-2-one;6-Hexyl-7-hydroxy-3-[4-(4-phenoxy-phe-nyl)-thiazol-2-yl]-chromen-2-one;7-Hydroxy-3-[4-(4-phenoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;6-Hexyl-7-hydroxy-3-[4-(4-methoxy-phe-nyl)-thiazol-2-yl]-chromen-2-one;7-Diethylamino-3-(4-methyl-thiazol-2-yl)-chro-men-2-one;3-[4-(4-Bromo-phenyl)-thiazol-2-yl]-6-hex-yl-7-hydroxy-chromen-2-one;6-Hexyl-7-hydroxy-3-(2-phenyl-thia-zol-4-yl)-chromen-2-one,6-Hexyl-7-hydroxy-3-(4-phenyl-thiazol-2-yl)-chromen-2-one;7-Hydroxy-3-(4-phenyl-thiazol-2-yl)-chro-men-2-one;6-Hexyl-7-hydroxy-3-(4-methyl-thiazol-2-yl)-chromen-2-one;7-Hydroxy-3-(5-methyl-4-phenyl-thiazol-2-yl)-chromen-2-one;3-[4-(4-Chloro-4-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;7-Hydroxy-3-(5-methyl-4-p-tolyl-thiazol-2-yl)-chromen-2-one;6-Bromo-3-[4-(4-ethoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;3-[4-(4-Ethyl-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[4-(4-Chloro-phenyl)-thiazol-2-yl]-6-meth-oxy-chromen-2-one;3-[2-(3,4-Dimethoxy-phenyl)-thiazol-4-yl]-chro-men-2-one;3-[4-(4-Bromo-phenyl)-5-ethyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-(4,5-Dihydro-naphtho[1,2-d]thiazol-2-yl)-7-hy-droxy-chromen-2-one;7-Diethylamino-3-(4-phenyl-thiazol-2-yl)-chro-men-2-one;3-[4-(3,4-Dichloro-phenyl)-5-methyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[4-(4-Chloro-phenyl)-5-ethyl-thia-zol-2-yl]-7-hydroxy-chromen-2-one;3-(5-Ethyl-4-p-tolyl-thiazol-2-yl)-7-hy-droxy-chromen-2-one;7-Diethylamino-3-(2-phenyl-thiazol-4-yl)-chro-men-2-one;3-[4-(2,5-Dimethyl-phenyl)-5-ethyl-thiazol-2-yl]-7-hydroxy-chromen-2-one;3-[2-(4-Hydroxy-phenyl)-thiazol-4-yl]-chro-men-2-one;3-(5-Ethyl-4-phenyl-thiazol-2-yl)-7-hy-droxy-chromen-2-one;3-[2-(2,4-Dimethyl-phenyl)-thiazol-4-yl]-7-hy-droxy-chromen-2-one;3-[4-(3-Bromo-phenyl)-thiazol-2-yl]-8-meth-oxy-chromen-2-one;7-Hydroxy-3-[4-(3-methoxy-phenyl)-thia-zol-2-yl]-chromen-2-one;3-Benzothiazol-2-yl-7-hydroxy-chro-men-2-one;3-(5-Chloro-1H-benzoimidazol-2-yl)-8-meth-oxy-chromen-2-one;3-[5-(3-Fluoro-phenyl)[1,3,4]oxa-diazol-2-yl]-8-methoxy-chromen-2-one;3-Benzo[d]imidazo[2,1-b]thiazol-2-yl-chro-men-2-one;3-(7-Methoxy-benzo[d]imidazo[2,1-b]thia-zol-2-yl)-chromen-2-one;6-Chloro-3-(7-fluoro-benzo[d]imi-dazo[2,1-b]thiazol-2-yl)-chromen-2-one;3-Benzo[d]imidazo[2,1-b]thiazol-2-yl-6-hex-yl-7-hydroxy-chromen-2-one;6,8-Dichloro-3-(5-p-tolylamino-[1,3,4]thia-diazol-2-yl)-chromen-2-one;3-[5-(3-Chloro-phenyl)-[1,3,4]oxa-diazol-2-yl]-6-hexyl-7-hydroxy-chro-men-2-one;6-Hexyl-7-hydroxy-3-[5-(3-methoxy-phe-nyl)-[1,3,4]oxadiazol-2-yl]-chro-men-2-one;6-Hexyl-7-hydroxy-3-(7-methyl-imidazo[1,2-a]pyridin-2-yl)-chromen-2-one;6-Methoxy-3-[2-(4-methoxy-phenyl-amino)-thiazol-5-yl]-chromen-2-one; and6-Hexyl-7-hydroxy-3-[5-(3,4,5-trimeth-oxy-phenyl)-[1,3,4]oxadiazol-2-yl]-chromen-2-one.55. The composition or combination of claim 31, wherein the anti-EGFRtherapeutic antibody is cetuximab or an antigen-binding fragment thereofor panitumumab or an antigen-binding fragment thereof.
 56. Thecomposition or combination of claim 1, wherein the composition orcombination comprises: a) i)3-(1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one, ii)midostaurin or a salt or solvate thereof, iii) gilteritinib or a salt orsolvate thereof, or iv)3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide;and b) an anti-EGFR therapeutic antibody.
 57. The composition orcombination of claim 49, wherein the anti-EGFR therapeutic antibody iscetuximab or an antigen-binding fragment thereof or panitumumab or anantigen-binding fragment thereof.